Microtubules are essential architectural elements within axons and dendrites that also act as railways for organelle transport. Axonal microtubules are nearly uniformly oriented with their plus ends distal to the cell body, while microtubules in vertebrate dendrites are non-uniformly oriented. These patterns are established by motor proteins that transport microtubules with different orientations into each type of process. Cytoplasmic dynein transports many of the microtubules with plus-ends leading, with the remainder of the work left to a class of kinesins best known for their roles in mitosis. The bona fide transport of microtubules occurs only when the microtubules are relatively short, but the forces imposed by these motors act on microtubules of all lengths. The forces imposed on long microtubules are important for determining whether the axon (and presumably dendrites) grows or retracts as well as for steering it toward its target tissues. The overall goal of this grant proposal is to understand how the relevant motors are regulated so that they can perform these important functions. Such knowledge will be instrumental for developing strategies for augmenting regeneration of injured nerves and for correcting flaws in microtubule orientation that can cause traffic jams in degenerating axons. The first specific aim seeks to elucidate the polarity orientation of the short mobile microtubules in the axon in order to test the hypothesis that mal-oriented microtubules are transported back to the cell body as a means of preserving the microtubule polarity pattern of the axon. Additional experiments are aimed at identifying the motors that transport microtubules of each orientation in their respective directions. The second aim seeks to test the hypothesis that the mitotic motors act at the level of the cell body to impose a limit on the transport of plus-end-distal microtubules into the axon while at the same time driving minus-end-distal microtubules into dendrites. The third aim seeks to investigate a potential role for post-translational modifications of tubulin in regulating the interaction of the relevant motors with microtubules in different compartments of the neuron. The proposed studies will utilize novel live-cell imaging techniques on cultured neurons, as well as innovative cell biological approaches for manipulating the relevant motor proteins.